Summary We propose to develop and apply new solution NMR and biophysical experiments tailored to reveal structural and dynamic changes of GPCRs upon ligand interactions in order to elucidate mechanisms of transmembrane signaling. G protein-coupled receptors (GPCRs) play central roles in many biological processes. Importantly, many of them are major drug targets for fighting allergies, schizophrenia, hypertension, psychosis, cancer or neurogenic pain. After the initial X-ray structure of a GPCR was solved (Cherezov et al. 2007) approximately 30 unique GPCR crystal structures have been reported and ~ 100 structures with various ligands (Munk et al., 2016). All of these represent static states of different active and inactive receptors but are limited in elucidating the dynamic mechanisms of signaling which is thought to involve transitions between many different dynamic states. We propose an extensive mapping of the dynamic states of the transmembrane region of GPCRs using solution NMR spectroscopy. Here we initially focus on the neurotensin receptor NTR1. The 13-residue neurotensin peptide plays important roles in multiple diseases, such as Parkinson, schizophrenia, antinociception, and hypothermia and in lung cancer. It is expressed throughout the central nervous system and in the gut, where it binds to at least three different neurotensin receptors (NTRs). NTR1 and NTR2 are class A GPCRs whereas NTR3 belongs to the sortilin family. Most of the effects of neurotensin are mediated through NTR1, where the peptide acts as an agonist, leading to GDP/GTP exchange within heterotrimeric G proteins and subsequently to the activation of phospholipase C and adenylyl cyclase, which produce second messengers in the cytosol. We propose to map the multifaceted dynamic states of the evolved HTGH4 and TM86V forms of NTR1 as model systems for revealing mechanisms of allosteric GPCR signaling. The planned research is founded on our engineering of covalently circularized nanodiscs, which can enclose and dramatically stabilize membrane proteins in patches of phospholipid bilayers (Nasr et al., 2017). Furthermore, we will rely on new advanced NMR, expression and labeling techniques that will allow extensive characterization of backbone end side chain dynamics. We will pursue three Specific Aims: Aim 1: Develop technologies for mapping the dynamic landscape of GPCRs in different ligand states. Aim 2: Achieve numerous backbone and side chain assignments for agonist-bound NTR1 in micelles and nanodiscs to obtain a dense network of probes sensing the dynamic state of the receptor. Aim 3: Characterize the ligand-free receptor, which is the main but least characterized state and reveal structural and dynamic changes upon binding agonists, antagonists and the heterotrimeric G protein Gi. Attempt NMR characterization of methyl signals of the heterotrimeric G protein.